doi: 10.1038/s41598-024-71540-x.
A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa
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- PMID:39227671
- PMCID: PMC11372047
- DOI: 10.1038/s41598-024-71540-x
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A user-friendly CRISPR/Cas9 system for mutagenesis of Neurospora crassa
Stefanie Grüttner et al. Sci Rep..
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Abstract
As a widely used eukaryotic model organism, Neurospora crassa offers advantages in genetic studies due to its diverse biology and rapid growth. Traditional genetic manipulation methods, such as homologous recombination, require a considerable amount of time and effort. In this study, we present an easy-to-use CRIPSR/Cas9 system for N. crassa, in which the cas9 sequence is incorporated into the fungal genome and naked guide RNA is introduced via electroporation. Our approach eliminates the need for constructing multiple vectors, speeding up the mutagenesis process. Using cyclosporin-resistant-1 (csr-1) as a selectable marker gene, we achieved 100% editing efficiency under selection conditions. Furthermore, we successfully edited the non-selectable gene N-acylethanolamine amidohydrolase-2 (naa-2), demonstrating the versatility of the system. Combining gRNAs targeting csr-1 and naa-2 simultaneously increased the probability of finding mutants carrying the non-selectable mutation. The system is not only user-friendly but also effective, providing a rapid and efficient method for generating loss-of-function mutants in N. crassa compared to traditional methods.
© 2024. The Author(s).
Conflict of interest statement
The authors declare no competing interests.
Figures
Schematic ofcas9 expression cassette integrated into the genome and verification of its integration, transcription and expression. (a) Illustration (not to scale) of the position of thecas9 expression cassette in the genome ofN.crassa strain #6103. (b) Illustration of thecas9 gene with oligonucleotides used for amplifying part of thecas9 sequence from genomic DNA and total RNA. Green arrow: forward oligonucleotide (SG3450); blue arrow: revers oligonucleotide (SR2883), (c) PCR amplifying part of thecas9 sequence andactin (as a control) from genomic DNA of the transformed #6103 strain (1–3). Genomic DNA of #6103 before transformation and water were used as negative controls. Verifying its integration into theN.crassa genome. Gel image has been cropped, for full size gel image see Supplementary Fig. 1. (d) RT-PCR amplifying part of the cDNA of thecas9 andactin (as a control) transcripts, using RNA of the transformants that were tested positive for thecas9 DNA sequence (1–3) and water as a negative control. For verification of thecas9 transcription. Gel image has been cropped, for full size gel image see Supplementary Fig. 1. (e) Immunoblot analysis of Cas9 expression of the Cas9-strain NcCas9SG using a Cas9 antibody. 17, 34, and 51 µg of total proteins were used in each lane, as indicated. The molecular weight of the respective protein is given in kDa. For loading control the ponceau-red stained membrane is shown. Image has been cropped, for full size membrane image see Supplementary Fig. 2.
Phenotypical comparison of NcCas9SG and #6103 (a) Comparison of aerial hyphae formation of NcCas9SG and #6103. VMM + S + his slant tubes were inoculated with 106 spores of the respective strain and after seven days at 25 °C growth was measured. The black line marks the starting point of the measurement. It was distinguished between the height up to where spore formation took place (white line) and the height of the growing hyphae (light blue). (b) Comparison of lateral hyphae growth of NcCas9SG and #6103. VMM + S + his plates were inoculated with 106 spores of the respective strain. After three days at 25 °C growth was compared.
Editing of thecsr-1 gene. (a) Thecsr-1 gene with exons (dark grey) and introns (light grey) is shown with the gRNA binding positions indicated in yellow (gRNA-c1) and blue (gRNA-c2); introns in light grey, exons in dark grey. (b) gRNA sequences. (c) Examples of sequence alignments ofcsr-1 sequences from edited fungi with the wild typecsr-1 sequence. Deletions are marked with a red and insertions with a purple line. Dots and green background indicate identical nucleotides, dashes indicate missing nucleotides, orange background indicates additional nucleotides.
Editing of thenaa-2 gene. (a) Thenaa-2 gene with exons (dark grey) and introns (light grey) is shown with the gRNA binding positions indicated in yellow (gRNA-n1) and blue (gRNA-n2). (b) gRNA sequences. (c) Examples of sequence alignments ofnaa-2 sequences from edited fungi with the wild typenaa-2 sequence. Deletions are marked with a red and insertions with a purple line. Dots and green background indicate identical nucleotides, dashes indicate missing nucleotides, orange background indicates additional nucleotides.
Comparison of different mutagenesis methods forN.crassa.
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